Metal chelators have been developed for the treatment of diseases resulting from metal overload. More recently, however, compounds capable of chelating iron are being studied as potential anticancer therapies, as iron has an important role in active sites of a wide range of proteins involved in energy metabolism, respiration, and DNA synthesis. One such protein, ribonucleotide reductase (RR) is an iron-containing protein that is essential for the conversion of ribonucleotides into deoxyribonucleotides for DNA synthesis and thus, a target for anti-cancer therapies. Many iron chelators are powerful inhibitors of RR due to their ability to bind iron (Richardson, D. R. (2002) Crit Rev Oncol Hematol 42(3): 267-81.). For example, the iron chelator desferrioxamine (DFO), which has been clinically approved for the treatment of iron overload diseases including β-thalassemia (Buss, J. L., B. T. Greene, J. Turner, F. M. Torti and S. V. Torti (2004) Curr Top Med Chem 4(15): 1623-35), has also been shown to be an inhibitor of RR. Moreover, some aggressive tumours have been shown to be sensitive to iron chelation by DFO. The use of DFO, however, is costly, requires long subcutaneous administration and the compound exhibits a short half-life. In addition to DFO, other iron chelators with anti-proliferative activity are in development, including Triapine (currently in phase II), 311, tachpyridine, and O-Trensox (Richardson, supra). Triapine may, however, have limited usefulness as an anticancer therapy as it exhibits low solubility in water.
U.S. Pat. No. 6,589,966 describes a novel family of metal chelators characterized as hexadentate chemical compounds that bind iron and that have antiproliferative activity against tumour cells. In addition, U.S. Patent Application No. 2002/0119955 describes additional compounds based on 3-AP (structurally related to Triapine) that may exhibit adequate therapeutic utility in the treatment of neoplasia, including cancer.
The chelation of zinc may also be an important but relatively unexplored determinant of the biological effects of iron chelators. For example, the iron chelator tachpyridine, which is under preclinical investigation as a potential anti-cancer agent, chelates zinc in addition to iron, and this may play a role in its cytotoxicity (Zhao, R., et al. (2004) Biochem Pharmacol 67(9): 1677-88.). Zinc has catalytic and structural roles in hundreds of zinc-dependent enzymes and zinc-finger motifs of proteins involved in DNA-protein or protein-protein interaction. Consequently, deficiency as well as overload of zinc causes a wide variety of alterations in mammalian metabolism. Depletion of zinc in vitro has been shown to cause apoptosis (McCabe, M. J., Jr., S. A. Jiang and S. Orrenius (1993) Lab Invest 69(1): 101-10), to significantly decrease cell proliferation of colon carcinoma HT-29 cells (Kindermann, B., F. Doring, M. Pfaffl and H. Daniel (2004) J Nutr 134(1): 57-62), and to alter cell cycle progression (Chen, X., et al. (2001) J Biol Chem 276(32): 30423-8.).
Alternatively, other metal chelators may exert anti-neoplastic effects through the formation of cytotoxic chelate complexes. This occurs predominantly with the redox-active metals, iron and copper. For example, bleomycins are a family of glycopeptide antibiotics with anti-tumour activity. They are used clinically in combination chemotherapy against lymphomas, squamous cell carcinomas and germ cell tumours. They contain a DNA binding domain and a metal binding domain, which binds Fe(II) or Cu(I). The presence of oxygen and a reductant leads to DNA cleavage through the formation of radical intermediates (Chen, J. and J. Stubbe (2005) Nat Rev Cancer 5(2): 102-12). The iron chelator Triapine, which inhibits RR by chelating iron, may also damage RR and other vital molecules by the generation of free radicals upon formation of the iron complex (Chaston, T. B., et al. (2003) Clin Cancer Res 9(1): 402-14). The use of bleomycin conjugates for targeting a compound to a body tumour are described in U.S. Pat. No. 4,758,421.
The cytotoxicity of the metal chelator 1,10-phenanthroline (OP) has been attributed to its ability to function as both chelator and chelate type. As a chelator, it has been shown to combine with zinc or iron and thus inhibit enzymes that require zinc or iron for activity. Alternatively, chelate complexes of 1,10-phenanthroline with divalent metal ions are reported to be cytotoxic (Shulman, A. and G. A. Laycock (1977) Chem Biol Interact 16(1): 89-99.) and the copper-chelate promotes the degradation of DNA (Downey, K. M., B. G. Que and A. G. So (1980). Biochem Biophys Res Commun 93(1): 264-70). Complexes of copper-OP can bind non-covalently to the DNA minor groove, and catalyze the single strand cleavage of nucleic acids in the presence of hydrogen peroxide and a reductant (Sigman, D. S., et al. (1979) J Biol Chem 254(24): 12269-72).
Copper-OP complexes are frequently used as chemical nucleases, and high-specificity DNA cleavage agents have been generated by attachment to sequence specific DNA binding proteins (Pan, C. Q., R. Landgraf and D. S. Sigman (1994) Mol Microbiol 12(3): 335-42.). OP is also used widely as an inhibitor of matrix metalloproteases (Springman, E. B., et al. (1995) Biochemistry 34(48): 15713-20) and has been shown to inhibit the synthesis of glycophosphatidylinositol (GPI) anchors (Mann, K. J. and D. Sevlever (2001) Biochemistry 40(5): 1205-13) through chelation of zinc.
Deregulation of tumour suppressor genes has been implicated in the development of cancer but the precise role of these tumour suppressor genes in the development of cancer is still not clear. The Krüppel-like factor (KLF) family of genes is a family of evolutionarily conserved zinc-finger containing transcription factors with diverse regulatory roles in cell growth, proliferation, differentiation and embryogenesis (Ghaleb, A. M., et al. (2005) Cell Res 15(2): 92-6). KLFs can function as either transcriptional activators or repressors or both, depending on their interaction with co-activators or co-repressors via specific amino-terminal domains, the promoters they bind, and the cellular context of their function (Kaczynski, J., T. Cook and R. Urrutia (2003) Genome Biol 4(2): 206). Several members of the KLF family are thought to be tumour suppressors and are involved in carcinogenesis. For example, down-regulation of KLF4 is found in colon cancer (Dang D T, et al. (2000) FEBS Lett, 476: 203-7) and down-regulation of KLF5 and KLF10 occurs in breast cancer (Chen C, et al. (2002) Oncogene, 21: 6567-72; Subramaniam M, et al. (1998) J Cell Biochem, 68: 226-36). KLF6 has also been suggested to be a candidate tumour suppressor gene at chromosomal location 10p15, with frequent mutations observed in prostate adenocarcinoma. Moreover, KLF6 was also shown to transactivate WAF1, which encodes a cyclin-dependent kinase inhibitor of the cell cycle via a p53-independent pathway (Narla G, et al. (2001) Science, 294: 2563-6).
Deregulation of KLF4 has been linked to cancers other than colon cancer both in vitro and in vivo, suggesting that KLF4 may have a tumour suppressor effect. In colorectal cancers, the level of KLF4 mRNA is reduced compared to normal matched tissues (Dang et al. (2000), supra), and re-expression of KLF4 in a colorectal cancer cell line results in diminished tumourigenicity (Dang, D. T., et al. (2003) Oncogene 22(22): 3424-30). A similar down-regulation and growth suppressive effect of KLF4 has also been described in bladder cancer (Ohnishi, S., et al. (2003) Biochem Biophys Res Commun 308(2): 251-6.), gastric cancer (Wei, D., et al. (2005) Cancer Res 65(7): 2746-54.), esophageal cancer (Wang, N., et al. (2002). World J Gastroenterol 8(6): 966-70), and adult T-cell leukemia (Yasunaga, J., et al. (2004). Cancer Res 64(17): 6002-9). In contrast to the tumour suppressor effect of KLF4, increased expression of KLF4 has been reported during progression of breast cancer (Foster, K. W., et al. (2000). Cancer Res 60(22): 6488-95.) and squamous cell carcinoma of the oral cavity (Foster, K. W., et al. (1999). Cell Growth Differ 10(6): 423-34.). In addition, KLF4 has been considered as a marker of an aggressive phenotype in early-stage infiltrating ductal breast carcinoma (Pandya, A. Y., et al. (2004). Clin Cancer Res 10(8): 2709-19.). Thus, while KLF4 likely plays a tumour suppressor role in gastrointestinal cancers and leukemia, the role of KLF4 in the development of other types of cancers is still not clear.
The expression of KLF4 is negatively regulated by zinc. Studies on the effect of zinc depletion on gene expression in colon carcinoma HT-29 cells using human oligonucleotide arrays showed that KLF4 gene expression was one of the most significantly up-regulated among ˜10,000 target genes tested. It has been hypothesized, therefore, that KLF4 may be a direct link between cellular zinc status and growth inhibition (Kindermann, B., F. Doring, M. Pfaffl and H. Daniel (2004). J Nutr 134(1): 57-62.). In a subsequent study, expression of KLF4 was found to be increased in cells over-expressing metal transcription factor-1 (MTF-1) (Kindermann, B., F. Doring, J. Budczies and H. Daniel (2005). Biochem Cell Biol 83(2): 221-9.). MTF-1 is a zinc-sensory transcriptional activator with six zinc-fingers, which binds to metal-responsive elements (MREs) of target genes, and the promoter of KLF4 also has 3 MREs. MTF-1 is usually up-regulated in zinc deficient cells and increased expression of MTF-1 has been observed in zinc-deficient HT-29 cells (Kindermann et al. 2004, supra). Therefore, zinc responsiveness of KLF4 in HT-29 is mediated at least in part by MTF-1 (Kindermann et al. 2005, supra). The expression of KLF4 is primarily associated with a terminally differentiated state of epithelial cells in organs such as gut, skin and thymus (Kaczynski et al. 2003, supra).
As described above, 1,10-phenanthroline (OP) is a well known metal chelator. Recent studies have investigated derivatives of 1,10-phenanthroline and their ability to chelate various metals. For example, Chao et al., have synthesized 1,3-bis([1,10]) phenanthroline-[5,6-d]imidazol-2-yl)benzene (mbpibH2) and its (bpy)2Ru2+ complexes and studied their electrochemical and spectroscopic properties (Polyhedron, 2000, 1975-1983). Liu et al., prepared ruthenium complexes with 2-(2-hydroxyphenyl)imidazo[4,5-f][1,10]phenanthroline (HPIP) and studied the binding behaviour of these complexes towards calf thymus DNA (JBIC, 2000, 5, 119-128). Similarly, Xu et al., have described the synthesis of 2-(4-methylphenyl)imidazol[4,5-f]1,10-phenanthroline and its Ru(II) complexes and binding of the prepared complexes to calf thymus DNA (New J. Chem., 2003, 27, 1255-1263).
International Patent Application No. PCT/IB04/052433 (WO 2005/047266) describes a broad class of 2,4,5-trisubstituted imidazole compounds, including some 1,10-phenanthroline substituted compounds, and their use in the treatment of cancer.
This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention.